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Myelodysplastic Syndromes

Alan F. List, James Vardiman, Jean-Pierre J. Issa, and Theo M. DeWitte

The development of new therapeutic strategiesfor myelodysplastic syndromes (MDS) hasgained new momentum fueled by improvedcharacterization of the disease’s natural historyand biology and by the recent US Food and DrugAdministration (FDA) approval of the first agentwith an indication for MDS. By integratingmorphologic and cytogenetic features withgreater discriminatory power, the World HealthOrganization (WHO) has refined the classificationof these stem cell malignancies and enhanced itsprognostic utility. Recognition that the malignantphenotype, which characterizes MDS, may arisefrom mechanistically diverse biological pro-cesses has raised new awareness that treatmentstrategies must be tailored to the pathobiology ofthe disease. Therapeutics targeting chromatinstructure, angiogenesis and the microenviron-ment that nurtures the MDS phenotype havedemonstrated remarkable activity and offer anopportunity to alter the natural history of the

disease. This chapter provides an overview ofrecent developments in the characterization ofMDS from the microscope to the laboratory andthe translation of these findings into promisingtherapeutics.

In Section I, Dr. James Vardiman reviews thecytogenetic abnormalities that characterize MDS,their clinical and pathologic significance, and theapplication of the WHO classification. In SectionII, Dr. Alan List reviews treatment goals driven byprognostic variables and biological features ofthe disease that have led to promising smallmolecule, selective therapeutics. In Section III,Dr. Jean-Pierre Issa provides an overview ofepigenetic events regulating gene expression,which may be exploited therapeutically bychromatin remodeling agents. In Section IV, Dr.Theo DeWitte discusses new developments inhematopoietic stem cell transplantation, includ-ing reduced-intensity and myeloablative ap-proaches.

I. CHARACTERIZATION AND CLASSIFICATION OF

MYELODYSPLASTIC SYNDROMES—FROM MORPHOLOGY TO CYTOGENETICS

James Vardiman, MD*

Although the myelodysplastic syndromes (MDS) wereinitially considered by many to be synonymous with“preleukemia,” this notion has given way to the realiza-tion that MDS is a heterogeneous spectrum of stem cellmalignancies, with the majority of patients succumb-ing to complications of bone marrow failure rather than

acute leukemia. In 1982 the French-American-British(FAB) cooperative group proposed morphologic guide-lines for the diagnosis and classification of MDS thatprovided a framework against which clinical, biologicand genetic studies could be universally compared.1 Sincethen nearly 20,000 publications have characterized vari-ous features of MDS. Despite such active investigation,a biologic marker that reliably identifies MDS remainselusive. Therefore, morphology remains the cornerstoneof diagnosis and an important tool that complementscytogenetic findings for prognostic discrimination.2

Nevertheless, particularly in “low grade” disease, themorphologic recognition of MDS can be difficult, cre-ating diagnostic indecision. For such cases, apprecia-tion of the basic guidelines for the interpretation ofmorphology and its correlation with clinical and cyto-genetic findings is essential for patient management.

* University of Chicago, 5841 S. Maryland Ave., MC0008,Chicago IL 60657

Acknowledgments: The author thanks Dr. Anna Porwit-MacDonald, Professor, Department of Pathology, KaronlinskaUniversity Hospitals, for her contribution of flow cytometryhistograms.

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Morphologic Characterization of MDS—Guidelines, Problems and Pitfalls

General guidelinesAlthough most hematologists and pathologists can re-cite the morphologic features of myelodysplasia, in prac-tice inter-observer reproducibility for recognition ofdysplasia is poor, particularly in low-grade MDS. Inone study, inter-observer agreement among five expertmorphologists was reasonably good for recognition ofblasts and ringed sideroblasts, but poor for dyserythro-poiesis (R = 0.27) and not much better for dysgranulo-poiesis (R = 0.45).3 A few simple guidelines can mini-mize such problems and improve recognition of MDS.

Prerequisites for evaluation of morphologic fea-tures in MDS include the availability of well-preparedperipheral blood and bone marrow aspirate smears. Theyshould be stained with Wright-Giemsa or May-Grunwald-Giemsa because Wright’s stain alone may notadequately demonstrate cytoplasmic granules.4 Ironstains of the marrow aspirate are essential to detect ringedsideroblasts. Blood and marrow smears should be ex-amined for dyplasia, the percentage of blasts and mono-cytes (nonspecific esterase stains may be helpful to de-tect monocytes in the marrow), and ringed sideroblasts.The enumeration of blasts is important for diagnosis,classification and prediction of prognosis.1,2 In myeloidneoplasms, myeloblasts, monoblasts, and megakaryo-blasts are included in the blast calculation. Small, dys-plastic megakaryocytes are not blasts and should not becounted as such. Erythroid precursors are also notcounted as blasts, except in rare cases of “pure” erythro-leukemia in which primitive erythroblasts account forthe majority of cells. In myelomonocytic proliferations,promonocytes are included as “blast equivalents.”4,5

Substitution of the percent of CD34+ cells determinedby flow cytometry for a visual blast count is discour-aged. Although hematopoietic cells that express CD34are blasts, not all blasts express CD34. In addition, di-lution of the marrow sample by peripheral blood dur-ing aspiration and processing of the sample for flowcytometry analysis complicates comparison between thevisual count and the CD34 value.

Although a bone marrow biopsy specimen is notalways necessary to establish a diagnosis of MDS, itoffers valuable diagnostic and prognostic information.4-6

Dysplasia, particularly of megakaryocytes, can be ap-preciated in well-prepared biopsies, and evidence ofdisruption of the normal marrow architecture, such asabnormal localization of immature precursors (ALIP),lends further support for the diagnosis of MDS. More-over, the biopsy provides confirmation of the blast per-centage and distribution, and serves as a source for im-

munohistochemical studies that may have diagnostic andprognostic value. An underappreciated role of the bi-opsy is that it may provide evidence for another diseasethat can mimic MDS clinically, such as lymphoma ormetastatic tumor. In cases of MDS that are hypocellularor associated with fibrosis, the biopsy is essential fordiagnosis.

Morphologic problems and pitfallsOne of the most difficult diagnostic challenges in MDSis that morphologic dysplasia is not specific for MDSbut can be seen in other conditions, including megalo-blastic anemia, congenital dyserythropoietic anemia,exposure to toxins such as arsenic and alcohol, aftercytotoxic and growth-factor therapy, and in HIV orparvovirus B19 infections—to name a few. Modest dys-erythropoiesis is also not uncommon when there is briskerythroid hyperplasia or regeneration, i.e., “stress eryth-ropoiesis.” This “secondary” dysplasia is most prob-lematic when only one (usually the erythroid ) lineageis involved, but multilineage dysplasia can also be atransient, reactive change. Causes of secondary dyspla-sia must be considered and excluded by appropriate clini-cal and laboratory studies prior to rendering a diagno-sis of MDS. Furthermore, a small number of dysplasticerythroid, granulocytic or megakaryocytic cells can beseen in marrow specimens from normal individuals.7

Hence, the guideline that 10% of the cells in a lineageshould be dysplastic to consider the lineage as dysplas-tic and as evidence for MDS is a reasonable rule ofthumb.6

The quality of the specimen is a common obstaclein the accurate diagnosis of MDS. For example, hypo-granularity of the cytoplasm of neutrophils is a well-accepted feature of dysplasia, but visualization of neu-trophil granules is critically dependent on an optimalstain. The diagnosis of MDS should never be based on“pale granulocytes” without other features to substanti-ate the diagnosis.6 Biopsies should be of adequate sizefor evaluation (at least 1–2 cm) and should extend intothe marrow well past the cortical bone. The marrowimmediately under the cortical bone is normally lesscellular than deeper marrow. In a case of MDS, thecombination of cytopenias in the blood and a superfi-cial, apparently hypocellular biopsy specimen mightresult in an erroneous diagnosis of aplastic anemia.

Even when these guidelines are carefully followed,the diagnosis of MDS may remain problematic becauseof morphologic features that are not clear-cut. Cytoge-netic studies may lend valuable support for the diagno-sis in such cases. In addition, characterization by flowcytometry may provide evidence for abnormal matura-tion of the myeloid lineages. Although no specific sur-

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face antigenic pattern is unique to MDS, the finding ofaberrant expression of antigens normally associated withdifferent stages of maturation of myeloid cells providesadditive information.8 In addition, abnormally granu-lated neutrophils may demonstrate abnormal light scat-ter properties on the flow cytometer (Figure 1; see ColorFigures, page 518). However, these abnormalities mustbe carefully interpreted in the light of other morpho-logical and clinical features because of their limitedspecificity.

Cytogenetic Characterization of MDSCytogenetic studies play a major role in confirmationof diagnosis and prediction of clinical outcome in MDS,and have contributed to the understanding of its patho-genesis. Clonal chromosomal abnormalities are detectedby routine karyotyping techniques in 40%–70% of casesof de novo MDS, and 95% of cases of therapy-relatedMDS (t-MDS).9 The relative frequency of the mostcommon cytogenetic abnormalities in de novo MDSare depicted in Figure 2. In t-MDS, deletion of part orall of chromosomes 5 and/or 7, and complex chromo-somal abnormalities account for 90% of the karyotypicchanges. If the morphology of a case is strongly suspi-cious but not entirely convincing for MDS, discoveryof a recurring chromosomal abnormality can lend strongsupport to the diagnosis. Occasionally, recurring cyto-genetic abnormalities are detected in cases suspectedclinically to be MDS because of unexplained cytope-nia, yet there is no morphologic dysplasia. In a recent

study of patients with these latter findings, Steensmaand associates reported that the cytopenia and chromo-somal abnormalities usually persist.10 They suggest thatin such cases the karyotypic abnormalities may be evi-dence of a “form fruste” of MDS and, in some cases,morphologic evidence of MDS will appear after vari-able length of follow-up.

The importance of cytogenetic abnormalities in theprediction of survival and in assessing the risk of trans-formation of MDS to acute leukemia is well known,and cytogenetic studies have been included in most ofthe predictive scoring systems developed for MDS.2,9

No cytogenetic abnormality is specific for MDS or fora specific morphologic subgroup of MDS. However,some unique cytogenetic/morphologic correlations ex-ist, the most common of which is the “5q- syndrome,”described in the classification section below.

Although it might be expected that chromosomalabnormalities would ultimately lead to the discovery ofthe genetic lesions important in the pathogenesis ofMDS, progress in this area has been slow. The patho-genesis of MDS is likely a multi-step process that in-volves a number of insults to the genome of a marrowstem cell, many of which are cytogenetically silent. Thesearch for genes critical to disease pathogenesis is fur-ther complicated because the characteristic chromosomalabnormalities in MDS involve loss of genetic material.Currently it is not clear whether the loss of geneticmaterial involves the total loss of function of a tumorsuppressor gene, a tumor suppressor gene that acts byhaploinsufficiency, or through some other associateddefect that has not yet been discovered. Gene expres-sion profiling of cDNA from patients with MDS hasrecently been reported. Genes reportedly upregulatedinclude those involved in cell proliferation, includingmembers of the Ras gene family, and some that aredownregulated, reportedly including genes encodinganti-apoptotic proteins.11

The WHO Classification of MDSThe FAB classification of MDS has been widely usedsince its introduction in 1982, and its clinical relevancehas been demonstrated in numerous studies. However,each FAB subgroup is heterogeneous and includes caseswith variable lineage involvement, various cytogeneticabnormalities, and widely variable clinical outcomes.In 2001, the World Health Organization (WHO) pub-lished new classification schemes for neoplasms of thehematopoietic and lymphoid tissues.12 For MDS, theWHO relied on data accumulated over the past two de-cades to propose modifications to improve the prog-nostic value of MDS classification. The major changesinclude (1) lowering the threshold for defining acute

Figure 2. Relative percentage of various cytogeneticabnormalities in de novo myelodysplastic syndrome(MDS).

Cytogenetic abnormalities are found in 40%–70% of patientswith de novo MDS and nearly 95% of patients with therapy-related MDS. This chart illustrates the relative distribution ofthe most common cytogenetic abnormalities in de novo MDS.

Percentage of WHO subtypes showing cytogenetic abnormali-ties: refractory anemia 24%, refractory anemia with ringedsideroblasts (RARS) 29%, refractory anemia with excessblasts–1 (RAEB-1) 35%, refractory cytopenia with multilineagedysplasia and ringed sideroblasts (RCMD-RS) 37%, refractoryanemia with excess blasts–2 (RAEB-2) 38%.

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myeloid leukemia (AML) from 30% to 20% blasts inthe bone marrow or peripheral blood with eliminationof the FAB category of refractory anemia with excessblasts in transition (RAEBT); (2) division of the low-grade categories of refractory anemia (RA) and refrac-tory anemia with ringed sideroblasts (RARS) into 5separate entities, depending on whether single lineageor multilineage dysplasia is present and on whether anisolated interstitial deletion of chromosome 5q is present;(3) subdividing RAEB into two categories dependingon the number of blasts in the blood and marrow; and(4) removing chronic myelomonocytic leukemia(CMML) from the MDS category into a new group ofdiseases, the Myelodysplastic/Myeloproliferative Dis-eases. The WHO classification and the criteria for eachsubgroup are shown in Table 1. The most controversialchanges in the WHO classification proved to be thereduction in the blast threshold for the diagnosis of AMLand the refinements in the FAB categories of RA andRARS, which deserve further comment.

Elimination of RAEBTA detailed discussion of the rationale for reducing thenumber of blasts required for the diagnosis of AMLand the elimination of RAEBT is found in reference 5.In brief, the WHO classification of AML includes anew subcategory, AML with multilineage dysplasia, thatis intended to capture cases of AML evolving from MDSor that have MDS-related features. A number of stud-ies have shown clinical, biologic and genetic similari-ties between the FAB category of RAEBT and the WHOcategory of MDS-related AML. Furthermore, 50%–60% of patients with RAEBT evolve to overt AMLwith 30% blasts or more within 6 months of their ini-tial diagnosis.2 An additional argument for the elimina-tion of RAEBT is that some patients with AML whohave no dysplastic-related features may have less than30% blasts on an initial marrow examination. Theywould therefore be assigned to the poor-risk categoryof RAEBT even though their leukemia is not MDS-related. Since publication of the WHO classification,

Table 1. The World Health Organization (WHO) classification of myelodysplastic syndromes (MDS).

Disease Blood Findings Bone Marrow Findings

Refractory anemia (RA) Anemia Erythroid dysplasia onlyNo or rare blasts < 10% grans or megas dysplastic< 1 × 109/L monocytes < 5% blasts

< 15% ringed sideroblasts

Refractory anemia with Anemia Erythroid dysplasia onlyringed sideroblasts (RARS) No blasts < 10% grans or megas dysplastic

≥ 15% ringed sideroblasts< 5% blasts

Refractory cytopenia with Cytopenias (bicytopenia or pancytopenia) Dysplasia in ≥ 10% of cells in two or moremultilineage dysplasia (RCMD) No or rare blasts myeloid cell lines

No Auer rods < 5% blasts in marrow< 1 × 109/L monocytes No Auer rods

< 15% ringed sideroblasts

Refractory cytopenia with multilineage Cytopenias (bicytopenia or pancytopenia) Dysplasia in ≥ 10% of cells in two or moredysplasia and ringed sideroblasts No or rare blasts myeloid cell lines(RCMD-RS) No Auer rods ≥ 15% ringed sideroblasts

< 1 × 109/L monocytes < 5% blastsNo Auer rods

Refractory anemia with excess blasts Cytopenias Unilineage or multilineage dysplasia– 1 (RAEB-1) < 5% blasts 5%–9% blasts

No Auer rods No Auer rods< 1 × 109/L monocytes

Refractory anemia with excess blasts Cytopenias Unilineage or multilineage dysplasia– 2 (RAEB-2) 5%–19% blasts 10%–19% blasts

Auer rods +/- Auer rods +/-< 1 × 109/L monocytes

Myelodysplastic syndrome, Cytopenias Unilineage gran or mega dysplasiaunclassified (MDS-U) No or rare blasts < 5% blasts

No Auer rods No Auer rods

MDS associated with isolated del (5q) Anemia Normal to increased megakaryocytes< 5% blasts with hypolobulated nucleiPlatelets normal or increased < 5% blasts

No Auer rodsIsolated del (5q)

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* H. Lee Moffitt Cancer Center and Research Institute,Malignant Hematology, SRB-4, 12902 Magnolia Drive, TampaFL 33612-9497

reports comparing survival of patients diagnosed asRAEBT by the FAB criteria with survival of patientsdiagnosed as MDS-related AML and 30% or more blastshave shown no significant differences.13,14 Admittedly,similar median survival times do not necessarily provesynonymy between the two groups.

Refractory anemia, refractory anemia with ringedsideroblasts, refractory cytopenia with multilineagedysplasia, and MDS with isolated del(5q) chromo-somal abnormalityThe FAB criteria for RA and RARS were vague as towhether these categories should include patients withdysplasia in lineages other than the erythroid lineageand, if so, how much dysplasia should be permitted. Inpractice, the FAB categories of RA and RARS wereheterogeneous and included MDS with unilineage eryth-roid dysplasia, as well as cases with severe multilineagedysplasia. A number of investigators have reported thatin low-grade MDS, multilineage dysplasia does influ-ence overall survival times and the incidence of trans-formation to acute leukemia.15,16 In the WHO classifi-cation, the criteria for RA and RARS were revised totake these studies into account. A new category, refrac-tory cytopenias with multilineage dysplasia (RCMD),was added to incorporate patients with MDS character-ized by fewer than 5% blasts in the marrow but dyspla-sia in 10% or more of the cells of at least two hemato-poietic lineages (see Table 1 for details). Additionally,the WHO recognizes another category of low-gradeMDS, the “5q- syndrome,” defined as having del(5q)as the sole chromosomal abnormality, < 5% blasts inthe marrow, and characteristic megakaryocytes withhypolobated nuclei.

Several studies have now reported that the WHOclassification does have improved prognostic value forthe low-grade MDS categories.17-19 In an analysis of103 patients diagnosed as RA by FAB criteria, Cermakand colleagues applied WHO guidelines (Table 1) toreassign 43 (42%) as “pure” RA, 56 (54%) as RCMD,and 4 (4%) as 5q- syndrome.18 For the entire group,median survival was 57.7 months, whereas for thosereclassified as “pure RA,” RCMD and 5q- syndrome,median survival times were > 102 months, 27 months,and 53.3 months, respectively. A separate report byHowe and associates showed that in a series of 64 pa-tients with low-risk MDS and < 5% marrow blasts,there was a significant difference in median survivalbetween patients with unilineage dysplasia (51% sur-viving at 67 mos) and those with multilineage dyspla-sia (median survival 28.5 mos).19 Recent clinical as wellas gene profiling studies have validated the WHO pro-posal that cases characterized by del(5q) as the sole cy-

togenetic abnormality comprise a unique group ofMDS.11,20

From the data currently available, it is likely theWHO classification will offer improved prognostic in-formation. Still, other categories of MDS are not ad-dressed by this classification, such as hypocellular MDSand MDS with fibrosis. These latter entities remainproblematic, and their recognition as well as their placeamong the other subcategories await further clarifica-tion. Perhaps, when it is time to revise the WHO classi-fication, these entities will be better understood and auniversally applicable biologic marker for MDS willbe available.

II. N OVEL THERAPEUTICS FOR AN ORPHAN DISEASE

Alan List, MD*

The remarkable hematologic, pathologic, and biologi-cal heterogeneity of the myelodysplastic syndromes(MDS) demands that disease-specific features be scru-tinized to properly gauge natural history of disease,determine therapeutic goals, and select optimal therapy.Morphologic classification alone is insufficient, deriv-ing prognostic power primarily from thresholds in blastpercentage.1,2 Prognostic modeling permits identifica-tion of variables with independent power for outcomeprediction. The International Prognostic Scoring Sys-tem (IPSS) represents the first such system to be ac-cepted worldwide for application in routine manage-ment decisions and clinical trials.3

The IPSS is derived from the analysis of data frommore than 800 patients with de novo MDS andnonproliferative CMML (i.e., WBC ≤ 12,000/µL) man-aged solely with supportive care. As such, expectationsfor survival and leukemia evolution reflect the intrinsicnatural history of disease that can serve as a benchmarkto be surpassed by novel therapeutics and managementdecisions. This model applies a score that is weightedaccording to the independent statistical power of eachof three prognostic features: bone marrow blast per-centage, cytogenetic pattern, and the number ofcytopenias (Table 2A). The cumulative score enablessegregation of patients into four subgroups with vary-ing expectations for survival, and risk of and intervalto AML progression (Table 2B). Although age offersfurther survival discrimination in lower risk patientsbecause of competing causes for death (i.e., Low- and

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Table 2B. Survival and risk of AML evolution by InternationalPrognostic Scoring System (IPSS) score.

IPSS Risk GroupLow Int-1 Int-2 High

Score 0 0.5–1.0 1.5–2.0 ≥ 2.5

Lifetime AML Evolution 19% 30% 33% 45%

Median Years to AML 9.4 3.3 1.1 0.2

Median Survival (years) 5.7 3.5 1.2 0.4

Table 3. Novel therapeutics categorized by pharmacologic class and targetof interaction.

Target and Pharmacologic Class Agent

Survival signals

Anti-Angiogenic thalidomide (Thalomid™)CC5013 (lenalidomide, RevliMid™)bevacizumab (Avastin™)arsenic trioxide (Trisenox™)

Receptor Tyrosine Kinase Inhibitors PTK787imatinib mesylate (Gleevec™)

Protein Kinase C Inhibitor PKC412

p38α MAPK Inhibitor SC10469

Matrix Metalloprotease Inhibitor AG3340 (Prinomastat™)

Farnesyl transferase Inhibitors R115777 (tipifarnib, Zarnestra™)SCH66336 (lonafarnib, Sarasar™)

Pharmacologic Differentiators TLK199 (Telintra™)

Table 2A. International Prognostic Scoring System (IPSS).

Prognostic variable 0 0.5 1.0 1.5 2.0

BM blast % < 5 5–10 — 11–20 21–30

Karyotype* Good Intermediate — Poor —

Cytopenias 0/1 2/3 — — —

*Good: normal, -Y, del(5q), del (20q); Poor: complex (≥ 3 abnormalities) orchromosome 7 anomalies; Intermediate: other abnormalities.

Adapted from Greenberg et al, International Scoring System for Evaluat-ing Prognosis in Myelodysplastic Syndromes.4

Intermediate-1 risk), it has no correlation with disease-related risks and therefore is not included in the prog-nostic model. The need to integrate morphologic andbiologic features with independent prognostic powerinto a clinically meaningful classification system thatcan be universally applied served as the impetus for theWHO’s recent proposals. Although this new schemaoffers greater prognostic discrimination than FAB, theIPSS complements both classification systems by itsincorporation of unfavorable cytogenetic abnormalitiesand number of lineage deficits.

These recently adopted tools for prognostic dis-crimination provided the foundation forcreation of universal measures of re-sponse by an International WorkingGroup (IWG).4 Like most other ma-lignancies, management must beguided by the risks imposed by the dis-ease, the patient’s age and performancestatus, expectation for treatment toler-ance, and quality of life. The therapeu-tic goals, therefore, should be judgedby the natural history of disease andpatient preference. Implicit in these rec-ommendations is the notion that pa-tients with Low- or Intermediate-1IPSS risk categories experience longersurvival, and therefore, amelioration ofhematologic deficits should representthe principal therapeutic goal and be

relatively durable to translate into clinicallymeaningful benefit. Such improvements there-fore must exceed minimally accepted thresh-olds for at least 2 month’s duration. For higher-risk patients (i.e., IPSS Intermediate-2/High riskcategories), extending survival is of immediatepriority, necessitating the incorporation of com-plete pathologic and cytogenetic remission asan early surrogate milestone for survival ex-tension.

Therapeutic DevelopmentsIdentification of transforming events integralto maintenance or propagation of the MDSclone has remained illusive. Nonetheless, de-lineation of biologic features that nurture theneoplastic phenotype has led to the develop-ment of novel therapeutics that have shown con-siderable promise. A partial summary of thoseagents in clinical development is provided inTable 3.

Angiogenic molecules generated by the neo-plastic clone represent one such lead that has

yielded promising new therapeutics for patients withhematologic malignancies. In MDS, in particular, vas-cular endothelial growth factor-A (VEGF-A) hasemerged as an important angiogenic molecule that isimplicated not only as a soluble effector of medullaryneovascularity but also in the clonal expansion of re-ceptor-competent myeloblasts as well as ineffective he-matopoiesis in receptor naïve progenitors.1,5 Paracrineinduction of inflammatory cytokines from receptor-competent adventitial cells within the microenvironmentpotentiates ineffective hematopoiesis by suppressingformation of VEGF receptor-naïve primitive progeni-

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tors. Based upon these and other preclinical investiga-tions, small molecule inhibitors of angiogenic cytokineshave emerged as a promising class of therapeutics forMDS with the principal impact on erythropoiesis.

Thalidomide (Thalomid™, Celgene Inc, WarrenNJ), which has both anti-angiogenic and tumor necro-sis factor (TNF)α inhibitory properties, represents thefirst agent in this class to be investigated in MDS. In aPhase II trial of thalidomide performed at the RushPresbyterian Cancer Institute,6 15 of 83 (18%) evaluablepatients experienced either red blood cell transfusionindependence or a > 50% decrease in transfusion bur-den, whereas improvement in non-erythroid lineageswas uncommon. Dose escalation beyond 200 mg dailywas limited by cumulative neurological toxicity and islikely unnecessary. An aggressive dose-escalationschema of 200 mg to 1000 mg daily evaluated by theNorth Central Cancer Treatment Group study was com-promised by excessive early attrition due to toxicity ata median interval of ≤ 2.5 months.7 Prolonged drugtreatment, when tolerated, appears necessary to maxi-mize hematological benefit. Median interval to eryth-roid response was 16 weeks in the Rush trial (range,12–20 weeks), with an erythropoietic response rate of29% among the 51 patients completing a minimum of12 weeks of study treatment. The overall clinical ben-efit of low-dose thalidomide in MDS was evaluated ina national randomized, placebo-controlled Phase III trialcompleted in the fall of 2003, the results of which shouldsoon be available.

Novel, more potent thalidomide analogues with im-proved toxicity profiles recently entered clinical inves-tigations.8 Lenalidomide (CC-5013, or Revlimid™:Celgene) is a 4-amino glutarimide derivative of thalido-mide that lacks the neurological toxicities of the parentcompound. It is a potent modulator of ligand-inducedcellular response with biological effects that range frompotentiation of antigen-initiated immune response, tomodulation of integrin affinity, suppression of trophicresponse to angiogenic molecules, and promoting pro-genitor responsiveness to erythropoietin.9 Among 36evaluable patients with MDS either with symptomaticor transfusion-dependent anemia treated with lenalido-mide in a safety and efficacy trial, 24 (67%) experi-enced an erythroid response according to IWG criteria,with 21 patients experiencing sustained transfusion in-dependence.10 Response rate varied by cytogenetic pat-tern and was highest among patients with a chromo-some 5q31.1 deletion (91%) compared to a normalkaryotype (68%) or other chromosome abnormality(17%) [P = 0.009]. Similarly, patients with lower riskIPSS categories experienced a higher frequency oferythroid response compared to patients with higher

risk disease (72% vs 25%); however, few patients hadIntermediate-2 or High-risk disease (n = 4). Unlikecytokine therapy, cytogenetic remissions were common,with 65% of informative patients experiencing 50% orgreater reduction in abnormal metaphases, including10 (57%) complete cytogenetic remissions. Major cy-togenetic response occurred most commonly in patientswith a chromosome 5q31.1 interstitial deletion (9 of 11patients). Perhaps of greater importance, responses ap-pear durable. After a median follow-up of 81 weeks,median duration of transfusion-independence had notbeen reached (48+; range, 13+ to > 101 weeks) withmedian sustained hemoglobin of 13.2 g/dL (range, 11.5–15.8 g/dL). Neutropenia (67%) or thrombocytopenia(57%) > grade 3 NCI-CTC was the most common ad-verse event and was dose dependent, necessitating treat-ment interruption or dose-reduction in 61% of patients.Lenalidomide has completed multicenter Phase II trialsin transfusion-dependent patients with Low- or Inter-mediate-1 risk MDS and either chromosome 5q31.1deletion (n = 148) or other karyotypic abnormalities (n= 215). The results of the trial, if sufficiently encour-aging, are expected to undergo accelerated review bythe US Food and Drug Administration (FDA) and maysecure a new position in the management of ineffectiveerythropoiesis for patients with MDS.

Small molecule inhibitors of the VEGF receptortyrosine kinases (RTK) have had limited investigationin MDS. SU5416 (Sugen Inc, S. San Francisco, CA),represents the only agent of its class to complete PhaseII investigation. Like most RTK antagonists, specific-ity is relative, with activity extending to other type IIIreceptors such as those for the PDGFβ, FLT3, and c-kitligands. A multicenter trial involving patients withhigher-risk MDS or AML yielded minimal reductionin leukemia burden and a corresponding degree of he-matological benefit despite increased apoptotic indexin the myeloblast population.11,12 Clinical developmentof this agent was limited by its insolubility and require-ment for twice weekly intravenous administration. In-vestigation of the orally bioavailable analogue SU11248in patients with AML ended prematurely owing to lim-iting nonhematological organ toxicities.13 Despite thedisappointing early results of this class of agents inmyeloid malignancies, clinical investigation of potentand orally active receptor antagonists continues. TheCancer and Leukemia Group B (CALGB) is investigat-ing PTK787 (Novartis, East Hanover, NJ) in patientswith low- and higher-risk MDS using a once daily ad-ministration schedule.

Arsenic trioxide (Trisenox™, Cell TherapeuticsInc., Seattle, WA) has broad biological properties thatderive from its ability to bind covalently and deplete

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cellular sulfhydryl-rich proteins such as glutathione, aswell as anti-angiogenic properties. Arsenic in its triva-lent form inhibits glutathione peroxidase to potentiateperoxide generation, disrupt mitochondrial respirationand mitochondrial membrane integrity, repress anti-apoptotic proteins and initiate caspase-mediatedapoptotic response.14 In MDS and AML, the anti-proliferative effects of ATO relate in part to its abilityto suppress myeloblast elaboration of VEGF-A and itsdirect cytotoxicity to neovascular endothelium.15 Notsurprisingly, bone marrow specimens from patients withMDS, which natively harbor lower glutathione reservescompared to normal hematopoietic progenitors,16 alsodemonstrate increased apoptotic susceptibility to ATOthat is enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation.17

Preliminary results of three clinical trials indicatethat ATO has modest activity in both lower- and higher-risk MDS.18-20 The doses and schedules applied in thesestudies vary, ranging from monthly cycles of two se-quential weekly treatments of 0.25 mg/kg/day for 5days followed by a 2-week treatment hiatus to a dose-intense induction with 0.30 mg/kg/day for 5 days fol-lowed by 0.25 mg/kg/day twice weekly maintenancefor 15 weeks. Overall, approximately 20%–25% of pa-tients have experienced hematological improvement,with few complete or partial remissions. Although eryth-roid responses predominate, hematologic benefit is notlimited to the erythroid lineage and responses may besustained for prolonged periods after treatment cessa-tion. Given the manageable toxicity of ATO, combina-tion trials are in progress, which may build upon theresults obtained with monotherapy.

Other anti-angiogenic agents investigated in MDShave demonstrated either a more limited toxicity:benefitprofile for extended use or have not as yet completedclinical investigation. Bevacizumab (Avastin™, Genen-tech, S. San Francisco, CA), a recombinant humanizedmonoclonal antibody that neutralizes VEGF-A in vivo,is currently completing Phase II investigation in MDS.21

Farnesyl Transferase InhibitorsActivating point mutations of the RAS proto-oncogeneare detected in fewer than 20% of unselected patientswith MDS but are common in CMML.1 The RAS genesuperfamily encodes guanosine triphosphate hydrolases(GTPase) that serve as critical regulatory elements insignal transduction, cellular proliferation and mainte-nance of the malignant phenotype. Farnesylation ofcarboxy-terminal consensus sequences by farnesyl pro-tein transferase (FPT) represents the first and rate lim-iting post-translational modification of Ras-GTPasesthat is requisite for membrane association and trans-

forming activity.22

The farnesyl transferase inhibitors (FTI) representa novel class of potent, oral inhibitors of Ras and otherprenylation-dependent proteins. These agents are ableto modulate multiple signaling pathways that have beenimplicated in the pathobiology or progression of CMMLand MDS in the absence of salvage isoprenylation path-ways. Preliminary results of Phase I/II studies in MDSand CMML indicate promising hematopoietic promot-ing activity that extends to non-erythroid lineages.23-25

Tipifarnib (R115777, Zarnestra ; Janssen Pharmaceu-ticals, Beerse, Belgium, and Spring House, PA) andlonafarnib (SCH66336 or Sarasar ; Schering-PloughResearch Institute, Kenilworth, NJ) are the leading non-peptide, heterocyclic oral FTIs that have completedPhase I and II clinical studies in hematological malig-nancies.23-25 In Phase I and II trials of tipifarnib per-formed in patients with MDS or AML, treatment with300–600 mg bid for 21 days every 4–6 weeks, yieldedpartial or complete responses in 20%–30% of patients,without relation to RAS mutation status.23,24,26 Interimresults of a multicenter Phase II trial involving patientswith either advanced MDS or elderly patients with AMLwho were not candidates for conventional chemotherapyinduction showed promising clinical benefit. Among98 evaluable patients treated with 600 mg bid for 21days every 4–6 weeks, 21% achieved a complete re-mission (CR), whereas 44% experienced either a CR,partial remission or hematologic improvement with amedian duration or remission approaching 5.5 months.26

Given the favorable treatment-related mortality (7%)compared to induction chemotherapy, this novel classof agents may create a new paradigm for the treatmentof advanced MDS and AML in the elderly. Importantly,the activity of this class of agents cannot be ascribedsolely to the inhibition of constitutively active RASproteins.

Efficacy and safety studies of lonafarnib adminis-tered in a continuous schedule have shown a compa-rable frequency of hematologic improvement but asomewhat lower apparent frequency of CR.25 Toxicityprofiles also differ with diarrhea and hypokalemia lim-iting at 300 mg twice daily. In an expanded Phase IItrial in 67 patients with MDS or CMML, erythroid re-sponses were reported in 35%, platelet responses in 22%of thrombocytopenic patients, and a 50% or greaterreduction in blast percentage was observed in 43% ofpatients with excess blasts. A Phase III randomized trialis planned to investigate the clinical benefit and fre-quency of platelet response to lonafarnib in patientswith CMML or advanced MDS with severe thromb-ocytopenia. Interestingly, 3 patients with proliferativeCMML (WBC > 12,000/µL) experienced rapid and sus-

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* University of Texas M.D. Anderson Cancer Center, Dept. ofLeukemia, 1515 Holcombe Blvd., Box 428, Houston TX 77030

The author wishes to thank Drs. Guillermo Garcia-Manero andHagop M. Kantarjian for their significant contributions to thispaper.

tained leukocytosis, which in 2 cases was complicatedby pulmonary infiltrates that resolved either after studydrug withdrawal or treatment with dexamethasone.27

The latter findings closely resemble the leukemia dif-ferentiation syndrome reported with retinoid therapyfor APL and may be linked to the unique ability oflonafarnib and perhaps other FPT inhibitors to activateβ-1 and β-2 integrins and promote both heterotypic andhomotypic adhesion of CMML cells.28 Overall, the fre-quency of leukemoid response to lonafarnib treatmentwas higher in patients with proliferative (≥ 12,000/µL)compared to nonproliferative CMML (54% versus 11%;P = 0.025). Close clinical monitoring of patients withproliferative variants receiving FTI treatment may bewarranted, with consideration for early introduction ofcytoreductive therapy.

Imatinib (Gleevec™)Constitutive Ras/mitogen-activated protein kinase(MAPK) activation is demonstrable in 40%–60% ofCMML cases, resulting either from mutations withinRAS alleles or from reciprocal translocations de-regulating RTKs.22,29 In the absence of mutations, sus-tained activation of the Ras/MAPK cascade may occurthrough a constitutive upstream signal. Perhaps the mostimportant therapeutic discovery in the management ofCMML in recent years is the activity of imatinib inpatients harboring a reciprocal chromosome transloca-tion involving chromosome 5q33. Although a numberof chromosomes and genes may partner in the generearrangements, the clinical phenotype is distinct, rec-ognized by the WHO classification as CMML with eosi-nophilia (CMML–Eos), but arising from the genera-tion of novel fusion genes involving the PDGFβ recep-tor with constitutive RTK signaling.1,30,31 Transgenicmouse models have shown that these novel RTK fusiongenes are singularly responsible for the generation ofthese myeloproliferative disorders and are selectivelyresponsive to PDGFβ kinase inhibitors.30 Imatinib bindsto the ATP-binding pocket of the PDGFβ receptor analo-gous to its interaction with BCR/ABL to act as a potentinhibitor of receptor kinase activity. Among 5 patientsreported to date, each achieved rapid hematologicalcontrol and sustained complete cytogenetic remissionwith imatinib monotherapy.31

Pharmacologic DifferentiatorsThe development of pharmacologic inducers of hemato-poietic differentiation in MDS has been limited by thechallenge of identifying relevant cellular targets whosefunction can be modified by synthetic small molecules.TLK199 (Telintra™, Telik, San Francisco, CA), a novelliposomal glutathione derivative that promotes granu-

lopoiesis both in vitro and in animal models, has re-cently entered investigations in MDS. TLK199 is thetripeptide diethylester, gamma-glutamyl ethyl ester (S-benzyl)cysteinyl-R(-)-phenylglycyl ethyl ester hydro-chloride and a selective inhibitor of glutathione S-trans-ferase P1-1 (GST P1-1), a member of a family of en-zymes that until recently were believed to exclusivelyfunction in cellular defense and drug detoxification.32,33

Recent investigations indicate that GST P1-1 is a nega-tive growth regulator, inhibition of which promotes theproliferation and differentiation of myeloid precursors.TLK199 undergoes intracellular de-esterification to theactive diacid form, TLK117, and is released to inhibitGST P1-1 and activate the MAPK pathway. This inhi-bition is believed to be responsible for its differentia-tion-promoting activity. Indeed, in animal models,TLK199 accelerates myeloid recovery from chemo-therapy-induced neutropenia as well as the myeloidgrowth factor G-CSF. Preliminary results of a Phase I/II trial in MDS have shown hematologic improvementin two or more lineages in 5 of 16 evaluable patients.34

While investigations with this agent continue, develop-ment of orally bio-available analogs is being explored.

III. C HROMATIN REMODELING AND EPIGENETIC

THERAPY IN THE MYELODYSPLASTIC SYNDROME

Jean-Pierre J. Issa, MD*

Chromatin remodeling is a powerful mechanism of regu-lating gene expression and protein function. In extremestates, chromatin remodeling can permanently repressexpression of a gene, a situation termed epigenetic si-lencing. Such silencing is exploited by cancers to fullyexpress the malignant phenotype. Reversal of silencing(epigenetic therapy) is an achievable goal in the clinic,and a promising new modality of treatment in MDSand other hematologic malignancies.

Chromatin Remodeling andthe Power of Epigenetics

We are what our genes say we are. This is, for the mostpart, true. However, an added level of complexity tothe physiologic functioning of multicellular organismsresides in chromatin control—specifically epigenetics.1,2

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DNA normally exists in a complex configuration withproteins such as histones. These protein-DNA interac-tions mediate packaging of DNA from ultra compact(the visible chromosomes during mitosis) to most re-laxed (the fine chromatin observed under the micro-scope in immature cells). The level of packaging helpsdetermine the expression status of DNA.

Epigenetics refers to stable changes in gene expres-sion that are mitotically stable and reversed only underspecial situations such as embryogenesis.1 Epigeneticsilencing, then, refers to nearly irreversible loss of geneexpression. This drastic mechanism of gene regulationis normally reserved for exceptional situations such asthe inactive X-chromosome in women and a few geneswhereby only one of the two copies of the gene is ex-pressed depending on the parent of origin, a processtermed imprinting. Epigenetic silencing, though usedfor a limited number of genes, is essential for the nor-mal development of mammalian cells.2

The mechanisms of chromatin remodeling for epi-genetic purposes have been the subject of intense inves-tigation. In mammals, two molecular mechanisms arekey to the process—DNA methylation and histone modi-fications.1,3 Methylation is mediated by the biochemi-cal addition of a CH3 group to various molecules. Meth-ylation can affect DNA, and the cytosine base is a spe-cific physiological target in mammalian cells. DNAmethylation plays important roles in development anddifferentiation and, over evolution, is thought to havebeen essential in suppressing the harmful effects of themyriad of retrotransposons (“jumping genes”) that lit-ter the human genome. Studies of the inactive X-chro-mosome established DNA methylation in promoter re-gions as key to maintaining epigenetic silencing. Thisis now thought to be achieved through tight interac-tions between DNA methylation and histone modifica-tions.2 This silencing cascade4 involves binding of me-thylated-DNA binding proteins (e.g., MeCP2) to themodified promoters, followed by recruitment of his-tone deacetylases, histone methylases, and eventually, asilencing complex of proteins including heterochroma-tin protein 1 (HP1). By this mechanism, chromatin isremodeled such that it becomes “invisible” to transcrip-tion factors, achieving a stable silenced state.

DNA Methylation in MDSEpigenetic processes, while required for development,are so drastic that they are not used for the dynamicregulation of gene expression. However, over the past15 years, it has become apparent that cancer cells usurpthe process of DNA methylation and use it to their ad-vantage by silencing the expression and function of genesthat counteract the malignant phenotype, such as tu-

mor-suppressor genes.5 Data from a variety of tumortypes has clearly established that gene silencing associ-ated with promoter DNA methylation is as powerful asgene mutations in functionally inactivating genes. It isused in cancer cells to affect most pathways requiredfor transformation such as proliferation, apoptosis, an-giogenesis, invasion, and immune evasion. Recent ex-periments have shown that epigenetic reprogrammingby nuclear transplantation erases the malignant pheno-type in some cell lines despite the persistence of geneticchanges,6 demonstrating that epigenetic abnormalitiesare full participants in malignant conversion.

Hematologic malignancies also demonstrate a linkbetween methylation and the neoplastic phenotype. Leu-kemias and MDS are characterized by the hyper-methylation and silencing of multiple genes.7 This pro-cess can occur early and has been detected in cases oflow-risk MDS but, in general, it is associated with dis-ease progression. In MDS, for example, the cyclin-de-pendent kinase inhibitor P15 is a frequent target of ab-errant methylation, and its inactivation is associated withan increased risk of progression to AML.8 A number ofother genes are similarly affected, included CDH1,CDH13, RIL, and others. There are data suggestingthat aberrant methylation is associated with resistanceto chemotherapy in AML9 and acute lymphocytic leu-kemia (ALL),10 and it is likely to play a similar role inMDS.

DNA Methylation InhibitorsThe discovery that hypermethylation contributes to themalignant process has rekindled interest in DNA me-thylation inhibition as a therapeutic strategy (“epige-netic therapy”) in cancer.11 Two cytosine analogs, 5-azacytidine (AZA) and 5-aza-2′ -deoxycytidine (DAC)were found 25 years ago to specifically inhibit DNAmethylation by trapping DNA-methyltransferases(MTases).12 AZA can incorporate into RNA and also isa pro-drug of DAC. DAC is phosphorylated by deoxy-cytidine kinase and incorporates efficiently into DNA.MTases, upon encountering DAC, form irreversiblecovalent bonds with the incorporated base and are thentargeted for degradation in the proteosome. Cells thendivide in the absence of MTases, which results in pro-gressive DNA hypomethylation and reactivation of pre-viously silenced genes. The covalent binding of MTasesto DNA can also result in cytotoxicity at high doses ofDAC and/or high levels of MTases.13 Both AZA andDAC were found to be active in vitro against a varietyof transformed cell lines, with particular efficacy inhematologic malignancies.

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AZAClinical trials with azacytidine were initiated two de-cades ago and revealed efficacy at high doses in acutemyelogenous leukemia and at lower doses in MDS.11 APhase III randomized study comparing AZA to sup-portive care in treatment-naïve MDS at various stagesdemonstrated response rates of 60% in the AZA arm(CR 7%, partial remission [PR] 16%, hematologic im-provement [HI] 37%) compared to 5% in the support-ive care arm (P < .001).14 These relatively durable re-sponses (median 15 months) translated into an improvedquality of life and a prolongation of median time toleukemic transformation or death from 13 months inthe supportive care arm to 21 months in the AZA arm.Side effects were relatively modest, consisting prima-rily of myelosuppression. These results led to the re-cent FDA approval of AZA for the treatment of MDS.

DACDAC is more active than AZA in vitro at equimolardoses and may have a different spectrum of activityand side effects compared to AZA because it does notincorporate into RNA.11 Clinical trials with DAC werealso initiated two decades ago and revealed promisingefficacy in hematologic malignancies. Phase II studiesof DAC in MDS revealed promising response rates ofaround 50% (CR rate around 20%), including cytoge-netic responses, and minimal nonhematologic toxicity.15

These results led to a multi-institution Phase III studyof DAC compared to supportive care in MDS, the re-sults of which will be announced at the 2004 ASH meet-ing. Data recently made public (but not subjected topeer review) suggested an improved time to AML ordeath in the DAC arm compared to the supportive carearm, particularly in treatment-naïve patients (354 daysvs 189 days, P = .03) and high-risk MDS (260 days vs79 days, P = .001).

Other DNA Methylation InhibitorsFavorable results with AZA and DAC led to intense re-cent interest in identifying additional MTase inhibitors,particularly orally available ones, or molecules that donot require DNA incorporation. A number of interestingapproaches have been described, including antisense andRNA interference approaches,16 the identification ofProcainamide as a weak MTase inhibitor,17 the suggestionthat a green tea component might inhibit DNA methyla-tion indirectly,18 and the recent discovery that Zebularine,an inhibitor of cytidine deaminase, also inhibits MTases.19

Clinical trials with these agents have either not shownpromising results or not been initiated yet.

Moving ForwardThe emerging field of hypomethylation therapy hasraised many interesting and occasionally crucial issuesin the treatment of malignancies.

DoseGiven that DAC has dual activity (hypomethylating atlow doses, cytotoxic at high doses), the issue of opti-mal dosing of this agent (or its congener AZA) needsto be reevaluated. Here, the classical maximally toler-ated dose (MTD) route to drug development is not in-dicated, and may have hindered the full evaluation ofthese drugs. Indeed, a recent study of low-dose DACreported favorable responses at doses 10–30 times lowerthan the MTD, with a suggestion of loss of response athigher doses.20 Also, the best reported responses withAZA and DAC are at relatively low doses, developedspecifically for the treatment of MDS, where most pa-tients are older and tolerate cytotoxic therapy poorly.Correlative studies suggest that the in vitro observationof rapid saturation of the hypomethylation effect (andloss of the differentiation effect) with increasing doses21

is also true in vivo. Current studies are exploring opti-mal dosing schedules for both AZA and DAC.

Mechanism of responseIt is not entirely clear whether responses to DAC andAZA are related to hypomethylation or cytotoxicity.The observation of decreasing responses with increas-ing dose favor hypomethylation,20 but this issue is farfrom completely settled. Moreover, even if hypo-methylation is the mechanism mediating responses,events downstream of hypomethylation remain to bedefined. Loss of methylation of the p15 tumor-sup-pressor gene was observed in patients with MDS treatedwith DAC,22 but p15 hypomethylation did not correlatewith response in a separate study.20 Possibilities includedirect cell death signaling by hypomethylation (per-haps through reactivation of retrotransposons), induc-tion of differentiation, induction of senescence, induc-tion of apoptosis through reactivation of proapoptoticmolecules,23 or immune responses through modulationof tumor antigens24 or even the host’s immune system.Pharmacodynamic studies of DAC in chronic myeloidleukemia (CML) suggest little effect in the first 5 daysof treatment and hypomethylation-related cell death inthe second week of therapy, but the mediators of thiseffect remain to be clarified.

CombinationsPerhaps the most exciting prospects of this field are theopportunities to improve clinical response by using com-binations of active drugs. These could be thought of in

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two broad categories. Combinations to improve epige-netic reactivation of silenced genes center on the mecha-nism of methylation-associated chromatin remodelingthat has been uncovered over the past few years. Thus,combinations of DAC and histone deacetylase inhibi-tors are synergistic in reactivating gene expression,25

and combinations with inhibitors of methylated-DNAbinding proteins or histone lysine 9 methyltransferasesare also attractive possibilities. Combination therapymay also be directed at optimally exploiting gene reac-tivation. Indeed, DAC has been shown in vivo to sensi-tize cells to the effects of biologic therapy such as retinoicacid26 and to increase the expression of pro-apoptoticmolecules,23 which may enhance the efficacy of classi-cal chemotherapeutic agents. It has also been demon-strated to reverse drug resistance in selected cases.27

Clinical trials exploiting combination epigenetic therapy(e.g., DAC and a histone deacetylase inhibitor such asvalproic acid) or making use of gene reactivation (e.g.,DAC and all-trans retinoic acid [ATRA]) are currentlyongoing.

IV. H EMATOPOIETIC STEM CELL TRANSPLANT

STRATEGIES IN PATIENTS WITH MYELODYSPLASTIC

SYNDROME AND SECONDARY ACUTE MYELOID

LEUKEMIA : THE ROLE OF REDUCED INTENSITY

CONDITIONING REGIMENS

Theo de Witte, MD, PhD*,and Margriet Oosterveld, MD

Allogeneic Stem Cell TransplantationThe primary curative treatment option for patients withMDS is allogeneic stem cell transplantation (SCT).Disease-free survival (DFS) ranges from 29% to 40%,with corresponding non-relapse mortality of 37% to50% and rate of relapse ranging from 23% to 48%with an HLA-identical sibling donor.1-5 Risk factorshaving an impact on the outcome of transplantation in-clude age, disease duration, disease stage at time of trans-

plantation, percentage of blasts in the bone marrow, thepresence of cytogenetic abnormalities, the source of stemcells, the application of T cell depletion of the graft,the type of donor and the intensity of the pretransplantconditioning.

In general the results of allogeneic SCT have im-proved in the past decade. The European Bone MarrowTransplant Group (EBMT) analyzed the treatment out-come of patients transplanted in three periods. The 3-year survival and DFS were better in patients trans-planted after 1989. This was due to a decrease in treat-ment-related mortality (TRM) over recent years.6 TheSeattle team recently reported favorable results in pa-tients with MDS treated with a busulphan-based regi-men in which the busulphan dosage was adjusted tomaintain blood levels at 800-900 ng/mL. The 3-yearnonrelapse mortality was 31% (28% related donors,30% unrelated donors) and relapse occurred in 16% ofthe patients with a related donor and 11% of the pa-tients with an unrelated donor.7

An EBMT survey in 234 patients with MDS com-paring marrow and G-CSF–mobilized peripheral bloodstem cells (PBSC) showed a lower treatment failurewhen PBSC were used as stem cell source.8 Use ofPBSC reduced the median duration of neutropenia andthrombocytopenia by 4 and 12 days, respectively, witha corresponding reduction in TRM (P = 0.007) exceptfor patients with RA. Chronic GVHD was more com-mon with PBSC (odds ratio: 1.62). The low treatmentfailure observed with PBSC in more advanced MDSstages suggests that a “graft-versus-MDS” effect existsand that it could be enhanced by the use of G-CSF-mobilized PBSC. The EBMT started a prospective studyto compare the value of PBSC versus bone marrowstem cells in April 2004. This study also addresses thequestion of whether remission-induction chemotherapyshould be administered to these patients prior to thetransplant conditioning.

Disease stage and agePatients with less advanced stages of MDS such as RAand RARS may profit optimally from allogeneic SCTwith a myeloablative regimen, with long-term DFS inmore than 50% of patients,9-10 owing largely to the sub-stantially lower relapse rate compared to patients withmore advanced disease.2

Longer disease duration before transplantation andolder age are associated with an increased risk of deathafter transplantation,10 thereby mandating considerationof transplant early in the course of the disease. Trans-plantation may be postponed in selected patients with-out life-threatening cytopenias and cytogenetic abnor-malities. A recent analysis by the Seattle group con-

* University Medical Center, Geert Grooteplein 8, PO Box9101, Nijmegen 6500 HB, The Netherlands

Acknowledgements: The authors wish to thank the physiciansand data managers of the transplant centers of the EuropeanBone Marrow Transplant Group (EBMT) for contributing theirdata and their experience. In addition they would like to thankthe EBMT data managers and statisticians for their analyses andsupport. Finally, they would like to thank the members of theMDS subcommittee of the Chronic Leukemia Working Party ofthe EBMT for their valuable contributions to the analyses andthe interpretation of the data.

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firmed that delayed transplantation may result in maxi-mized overall survival for low and intermediate-1 IPSSgroups.11 They hypothesized that the optimal timing oftransplantation for this cohort is at the time of develop-ment of a new cytogenetic abnormality, the appearanceof a clinically important cytopenia or an increase in thepercentage of marrow blasts.

Data on allogeneic SCT in CMML are limited.1,12,13

Prognostic modeling shows that marrow infiltration withmore than 5% monoblasts, a neutrophil count of morethan 16 × 109/L and/or a monocyte count of more than2.6 × 109/L are associated with an unfavorable prognosisand therefore patients with these features should be con-sidered for for allogeneic SCT. In an analysis of 50 CMMLpatients reported to the EBMT registry, the estimated 2-year DFS was 18% with a relapse risk of 42%.13

Outcome of SCT in patients with RAEB andRAEBt is less favorable than the outcome in patientswith RA(RS), due largely to a higher risk of relapse.The EBMT reported a 5-year actuarial relapse rate of44% and 52% in 35 RAEB patients and 28 RAEBtpatients.14 The Fred Hutchinson Cancer Research Cen-ter (FHCRC) reported a 49% relapse rate for patientswith excess of blasts compared to 4% for patients with-out marrow blast elevations,12 with actuarial DFS of31% versus 54%, respectively. Among 885 patientstransplanted with an HLA-identical sibling in the EBMTregistry, 3-year probability of DFS, overall survivaland relapse were 36%, 41% and 36% respectively.6 Bothage and disease stage had independent prognostic sig-nificance for all three end-points. A similar report bythe International Bone Marrow Transplant Registry(IBMTR) in 452 recipients of HLA-identical siblingtransplants performed between 1989 and 1997 confirmedthe influence of young age and low percentage of mar-row blasts at time of transplantation on high DFS andoverall survival rates.15

In patients with secondary AML (sAML) afterMDS, most European transplant centers have adoptedthe strategy of SCT after remission-induction chemo-therapy based on the high failure rate of SCT in pa-tients with active leukemia.2,3,16

Whether patients with advanced stages of MDS orsAML benefit from chemotherapy prior to transplanta-tion is still unresolved. The superior outcome for pa-tients with a lower blast percentage supports the use ofchemotherapy to lower the disease burden before trans-plantation. Only prospective randomized studies withanalyses based on the intention-to-treat principle willovercome the selection bias inherent in retrospectiveanalyses. As noted above, the EBMT has launched sucha study.

Cytogenetic abnormalitiesCytogenetic abnormalities have a major influence onthe outcome after SCT. A French study3 reported a 7-year relapse rate of 83% in patients with complexanomalies. Using cytogenetic risk categories definedby the IPSS, event-free survival for the poor-risk, in-termediate-risk and good-risk groups were 6%, 40%and 51%, respectively, with actuarial risk of relapse82%, 12% and 19%, respectively.17

Therapy-related MDS/AMLA recent report from French investigators involving 70patients with therapy-related MDS and AML18 included34% of patients in complete remission at the time oftransplantation. Two-year event-free survival, relapseand TRM rates were 28%, 42% and 49%, respectively.Only 5 of the 46 patients with active disease at the timeof transplantation were long-term survivors. A largestudy from Seattle reported on 99 patients (47 tMDS,52 tAML). Sixty-five patients received marrow from afamily member and 34 received marrow from an unre-lated donor. The probability of survival, relapse andnon-relapse mortality was 13%, 47% and 78%, respec-tively.19

T cell depletionT cell depletion did not influence outcome in a recentmultivariate analysis performed by the IBMTR despitean increased relapse risk.15 However, an earlier, singlecenter study, study showed a 73% DFS at 2 years aftertransplantation for RA with T cell–depleted grafts fromHLA-identical siblings using elutriation.20

Reduced-intensity conditioning regimensThe principle of reduced-intensity conditioning (RIC)is to minimize toxicity associated with conventionalmyeloablative regimens and harness the graft-versus-MDS effect of the infused donor lymphocytes. RIC regi-mens depend largely upon intensive immune suppres-sion either during conditioning and/or after stem cellinfusion to facilitate donor engraftment and establishcomplete donor chimerism. Kröger et al21 reported 37patients with MDS or secondary AML, half of whomhad a related donor, who were ineligible for conven-tionally conditioned transplants. The reduced-intensityconditioning consisted of fludarabine, busulphan andantithymocyte globulin. Overall TRM was 27%, withsignificantly higher mortality in those with poor-riskcytogenetics (75% vs 29%) or with an HLA-matchedunrelated donor (45% vs 12%). In total, 32% of pa-tients relapsed, and actuarial DFS at 3 years was 38%with a median follow-up of 20 months. A Spanish studyshowed a TRM of only 5% after transplantation of 37

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patients with MDS and AML (median age: 57 years)utilizing a regimen of fludarabine and busulphan 10mg/kg.22 The 1-year progression-free survival was 66%with a corresponding frequency of disease-progressionin patients with and without graft-versus-host disease(GVHD) of 13% (95% CI, 4%–34%) and 58% (95%CI, 36%–96%), respectively (P = 0.008). These resultssupport the notion that a graft-versus-MDS/AML re-sponse is critical in reducing the risk of relapse after anRIC transplant.22 Stuart et al23 described the results of91 patients with a diagnosis of MDS (n = 77) or MPDexcept CML (n = 14) who were conditioned withfludarabine and a single fraction of total body irradia-tion (2 Gy) followed by infusion of stem cells from anHLA-matched related (n = 49) or unrelated (n = 42)donor. Patients with low-risk MDS (RA, RARS, RAEB)at the time of transplant (n = 33) had an 18-month relapserate of 32% ± 18%, resulting in overall survival rates at18 months of 40% ± 18%. This relatively high relapserisk is in line with the observations of a recent EBMTstudy. The EBMT analysis showed a 54% relapse risk forthe 24 patients transplanted with RIC protocols, whichtranslated into an increased HR of 6.0 (P = 0.02) in themultivariate Cox model.24

The King’s College Hospital group from Londonreported more favorable results following conditioningwith fludarabine, busulphan and alemtuzimab(Campath-1H) in 62 patients with MDS (24 matchedsibling donors or 38 unrelated donors). One-year DFSwas 61% and 59% in patients transplanted with siblingand unrelated donors, respectively. The favorable re-sults may be explained by the low estimated 1-year TRMof 15% (5% sibling, 21% voluntary unrelated donors[VUD]), the relatively high number of patients trans-planted with less than 5% marrow blasts (> 75% of thepatients) at the time of transplant conditioning, the highnumber of patients who received donor lymphocyteinfusions (67% of sibling recipients and 26% of VUDrecipients) and the relatively short period of follow-up.25 No long-term surviving patients were observed inpatients with progressive disease.

Martino analyzed 196 cases of MDS reported tothe EBMT after transplantation with RIC regimens. Ina multivariate analysis the survival and DFS were notinfluenced by the type of conditioning despite an in-crease in the risk of relapse after RIC.26 It is difficult toreconcile the contribution of RIC regimens to the im-proved outcome of allogeneic SCT for patients withMDS in view of the recently improved outcome of trans-plantation with marrow ablative regimens and the het-erogeneity of the patient populations (age, co-morbid-ity, stage of disease). For this reason, the EBMT haslaunched a prospective randomized study comparing

RIC regimens with standard conditioning regimens inpatients with MDS older than 50 years for whom anHLA-identical sibling is available and in all age cat-egories for patients with potential unrelated donors.Patient accrual started in April 2004.

Transplantation with alternative donorsAmong patients with MDS transplanted at the FHCRCwith an unrelated donor following myeloablative con-ditioning, 2-year DFS was 38% with a relapse rate of28%, and non-relapse mortality of 48%.27 Both olderage and longer disease duration were associated with agreater risk of death from non-relapse causes. Among118 patients who received an SCT from an unrelateddonor in the EBMT database, DFS at 2 years, relapserisk and TRM were 28%, 35% and 58%, respectively.28

The TRM was significantly influenced by age (youngerthan 18 years: 40%; 18–35 years: 61%; older than 35years: 81%). Patients with more severe acute GVHDexperienced a lower relapse risk, suggesting an increasedgraft-versus-MDS effect in these patients. The Ameri-can National Marrow Donor Program (NMDP) reportedan improved DFS in more recent transplantations in acohort of 510 patients with MDS transplanted with un-related donors. The relative risk for DFS was 1.43 (95%confidence interval: 1.01–2.01) for transplantationsperformed between 1988 and 1993 versus more recenttransplantations.29

By comparison, among 91 patients transplanted withstem cells from genotypically non-identical related do-nors in the EBMT database,6 3-year DFS, survival andrelapse rate were 28%, 31% and 18%, respectively. Itis noteworthy that the TRM was 66%, higher than inany other type of transplantation. Table 4 shows a sum-mary of studies published in the past years on alloge-neic BMT for MDS and sAML.

Autologous Stem Cell TransplantationFor those patients lacking a suitable donor, intensivechemotherapy with AML-like schedules may be an alter-native approach. Complete remission rates have improvedin recent years, ranging between 15% and 65%.30-34 Re-mission duration, however, is brief due to the high rateof relapse. Karyotype is the most important prognosticfactor influencing DFS with a median of 16.5 monthsfor patients with a normal karyotype compared to 4months in those with an abnormal karyotype.32 In 1995the Leukemia Cooperative Group of the EuropeanOrganisation for the Research and Treatment in Cancer(EORTC) reported results of the first prospectivemulticenter study using cytarabine and idarubicin asremission-induction treatment in patients with high-riskMDS and sAML.33 There was difference in remission

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Table 4. Allogeneic stem cell transplantation for myelodysplastic syndrome (MDS) and secondary acute myeloidleukemia (sAML).

rates between patients with MDS (50%) and sAML pa-tients (63%), with outcome adversely affected by anabnormal karyotype. In an analysis of 158 patients withhigh-risk RAEB and RAEBt and 372 AML patientswith AML treated at the MD Anderson Cancer Center,remission rates were comparable for RAEB, RAEBtand AML, but EFS and overall survival were inferiorin RAEB compared to AML or RAEBt.35 Multivariateanalysis indicated that the poor outcome in this mor-phologic group was linked to disproportionate adverseprognostic features, in particular, complex cytogeneticabnormalities.

In view of the high relapse rate after chemotherapyalone, transplantation with autologous stem cells hasbeen applied in an attempt to intensify the postremissiontherapy. In 1997 the EBMT reported the results of 79patients autografted for MDS and sAML in first com-plete remission.36 Two-year survival, DFS and relapse

rate were 39%, 34% and 64%, respectively. Patientsyounger than 40 years showed a significantly better DFS(39%) than patients older than 40 years (25%). In 1999the first prospective study on autologous SCT in MDSwas published.37 A complete remission was attained in42/83 patients (51%). In 24 out of 39 patients (62%)transplantation with autologous bone marrow (ABMT:16 patients) or peripheral blood stem cells (APSCT: 8patients) was performed. Hematological reconstitutionoccurred in all autografted patients. However, this study,perhaps given its size limitations, did not confirm a fasterhematopoietic recovery for peripheral blood stem cellscompared to bone marrow. The median DFS of theautografted patients was 29 months from transplantation.

The results of several studies employing intensivechemotherapy with or without SCT are summarized inTable 5. A multicenter study of the EORTC, EBMT,the Swiss Group for Clinical Cancer research (SAKK)

Median OutcomeNumber of Age Calculated DFS or

Source Patients (years) at N yrs. EFS (%) R elapse (%) TRM (%)

HLA-Identical Sibling

Anderson 1995 12 93 30 5 40 29 44

Sutton 1996 3 71 37 7 32 48 39

Runde 1998 14 131 33 5 34 39 44

Nevill 1998 13 60 40 7 29 42 50

De Witte 2000 6 885 33 3 36 36 43

Sierra 2002 15 452 38 3 40 23 37

Deeg 2002 7 41 46 3 56 16 28

Anderson 1997 16 # 46 42 5 24 31 44

Yakoub-Agha 2000 18 # 70 37 2 28 42 49

Voluntary Unrelated Donor

Anderson 1996 27 52 33 2 38 28 48

Arnold 1998 28 118 24 2 28 35 58

De Witte 2000 6 198 — 3 25 41 58

Deeg 2002 7 64 46 3 59 11 30

Castro-Malaspina 2002 29 510 38 2 29 14 54

Reduced Intensity Conditioning

Martino 2002 26 37 57 1 66 28 5

Ho 2004 25 24 56 1 61 5

Ho 2004 25 (VUD) 38 52 1 59 21

# Transplantation for therapy-related MDS and AML (tMDS/tAML)

Notes: Nevill 1998: including 22 unrelated donors; Deeg 2002: age 46 yr overall; Anderson 1997: including 17 tAML and 29 sAMLpatients; Yakoub: including 8 unrelated donors, 3 mismatched related donors; Martino 2002: including 17 AML patients

Abbreviations: DFS, disease-free survival; EFS, event-free survival; TRM, treatment-related mortality

312 American Society of Hematology

and Gruppo Italiano Malattie Ematologiche dell’ Adulto(GIMEMA) compared the results on 159 patients whohad received remission-induction chemotherapy andthen were candidates for allogeneic and autologous stemcell transplantation depending on the availability of anHLA-identical sibling.38 Sixty-nine percent of the pa-tients with a donor underwent allogeneic SCT and 49%received an autograft. The 4-year EFS was 23% forpatients with a donor and 22% for patients without adonor (P = 0.66). This study suggests that patients withhigh-risk MDS and sAML may benefit from either al-logeneic or autologous SCT. The results of this studywere compared with the outcome of 215 MDS and MDS-AML patients treated at the MD Anderson Cancer Cen-ter.39 MDS patients had received varied high-dosecytarabine-containing induction regimens, and afterremission continued to receive these regimens at re-duced dosage for 6–12 months. Remission rates were54% and 63% respectively (P = 0.09). Sixty-five ofthe EORTC patients who entered CR received a trans-plant in first CR. DFS in patients achieving CR wassuperior in the EORTC cohort, the 4-year DFS rateswere 29% EORTC versus 17% MDA (P = 0.02), butthe survival was not significantly different between thetwo study groups.

Since MDS is a clonal stem cell disorder, there re-mains concern regarding contamination of the graft byresidual malignant cells, and residual normal stem cells

are sufficient to support rapid reconstitution. However,several studies reported that patients with an abnormalkaryotype can achieve a cytogenetic remission if amorphological remission is reached after chemotherapy.This is supported by murine models in which no clonal(cytogenetically aberrant) precursors were identified inNOD/SCID mice transplanted with marrow from patientswith MDS more than 2 months after transplantation.40

Delforge et al reported that polyclonal primitivehematopoietic progenitors can be mobilized in patientswith high-risk MDS after treatment with intensive che-motherapy.41 Clonality analysis was performed in fe-males heterozygous for the X-linked human androgen-receptor (HUMARA) gene demonstrating a polyclonalpattern in the CD34+ cell population in 4 of 5 patients.

In a separate report involving 11 patients in CRafter chemotherapy, stem cell mobilization was at-tempted either with G-CSF alone or with recovery fromconsolidation. In 7/11 patients CD34 cell yields exceeded1 × 106/kg,42 and karyotypically normal progenitors wererecovered exclusively in 6/9 patients who presented withan abnormal karyotype. In our own experience stem cellmobilization was feasible in about 50% of 24 patients inthe recovery phase after chemotherapy with G-CSF.

ConclusionsAllogeneic SCT is the treatment of choice for the ma-jority of young patients with MDS or sAML who have

Table 5. Intensive chemotherapy with or without autologous stem cell transplantation in myelodysplastic syndrome(MDS) and secondary acute myeloid leukemia (sAML).

Median Number ofNumber of Age Induction CR Patients

Source Patients (years) Chemotherapy (%) Transplanted Outcome #

Ossenkoppele 2004 30 MDS 91 AML 43 65/69 A + G-CSF + F 68 — DFS 23/16% OS 39/24%*

Fenaux 1991 32 MDS 31 sAML 16 54 Z + A 47 — DFS: 11 mo OS: 14 mo

De Witte 1995 33 MDS 34 sAML 16 46 I + A 54 — DFS: 11 mo OS: 15 mo

Parker 1997 34 MDS 13 sAML 3 44 I + A + F + G-CSF 63 6 ## Too short follow-up

Estey 1997 35 MDS 158 60 I + A or F + A ± G-CSF 65 — DFS : 5-12 mo(RAEB/RAEBt)

De Witte 1997 36 ### MDS 19 sAML 60 39 — 79 DFS: 34% OS: 39%relapse: 64%

Wattel 1999 37 MDS 37 sAML 46 45 A + M ± Q 51 24 DFS: 29 mo OS: 33 mo

Oosterveld 2003 38 MDS 91 sAML 28 47 A + I + E 54 32/65*** EFS: 23*

#: Outcome: median duration in months or percentage at 2 years or 4 years

##: 3 allogeneic BMT, 3 autologous stem cell transplantation###: Report on 79 patients transplanted in first CR

*: DFS and OS with or without fludarabine respectively

**: 65 patients without HLA-identical sibling donor

Abbreviations: CR, complete remission; A, cytarabine; Z, zorubicine; I, idarubicin; M, mitoxantrone; Q, quinine; E, etoposide; G-CSF,granulocyte colony-stimulating factor; DFS, disease-free survival; OS, overall survival; EFS, event-free survival; RAEB, refractoryanemia with excess blasts; RAEBt, RAEB in transformation

Hematology 2004 313

a histocompatible sibling. Long-term DFS can be at-tained if SCT is performed early in the disease course.Since transplant outcome is superior for patients with alow blast percentage, successful suppression of leuke-mia burden by chemotherapy may be justified prior toSCT in patients with advanced disease. A definitiveanswer to this long-standing question must await theresults of the EBMT trial. Transplant outcome for pa-tients with MDS remains inferior to that for de novoAML owing to the high TRM and rate of relapse. Sub-sequent studies must optimize selection schedule, con-ditioning regimens and posttransplant immune-modulation. Immunotherapy with donor lymphocyteinfusions has been successful in selected cases of re-lapse after allogeneic SCT.43-45s RIC regimens allow al-logeneic transplantation in recipients of older age orwith co-morbidity, a frequent reality in the treatmentof patients with MDS. Moreover, RIC allows optimalutilization of posttransplant immunomodulation withdonor lymphocyte transfusions. However, the place ofRIC remains to be determined since the results of con-ventional, bone marrow ablative regimens have im-proved in recent years. Prospective, randomized stud-ies, such as that initiated by the EBMT, are necessary toelucidate the contribution of RIC regimens to the treat-ment of MDS patients.

For patients lacking an HLA-identical sibling, theoutcome with autologous SCT appears comparable toallogeneic transplantation with donors other than HLA-identical siblings and phenotypically identical familymembers with lower TRM. Achievement of completeremission and harvest of a sufficient number of autolo-gous stem cells are prerequisites for autologous SCT.For patients who fail to achieve remission, allogeneicSCT with unrelated donors remains an alternative foryounger patients.

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